Webb, S.M., G.J. Dick, J.R. Bargar, and B.M. Tebo. Evidence for the presence of Mn(III) intermediates in the bacterial oxidation of Mn(II) (2005). Proceedings of the National Academy of Sciences, 102: 5558-5563. (abstract) (.pdf)

Metagenomics has provided access to genomes of as yet uncultivated microorganisms in natural environments, yet there are gaps in our knowledge—particularly for Archaea—that occur at relatively low abundance and in extreme environments. Ultrasmall cells (<500 nm in diameter) from lineages without cultivated representatives that branch near the crenarchaeal/euryarchaeal divide have been detected in a variety of acidic ecosystems. We reconstructed composite, near-complete ~1-Mb genomes for three lineages, referred to as ARMAN (archaeal Richmond Mine acidophilic nanoorganisms), from environmental samples and a biofilm filtrate. Genes of two lineages are among the smallest yet described, enabling a 10% higher coding density than found genomes of the same size, and there are noncontiguous genes. No biological function could be inferred for up to 45% of genes and no more than 63% of the predicted proteins could be assigned to a revised set of archaeal clusters of orthologous groups. Some core metabolic genes are more common in Crenarchaeota than Euryarchaeota, up to 21% of genes have the highest sequence identity to bacterial genes, and 12 belong to clusters of orthologous groups that were previously exclusive to bacteria. A small subset of 3D cryo-electron tomographic reconstructions clearly show penetration of the ARMAN cell wall and cytoplasmic membranes by protuberances extended from cells of the archaeal order Thermoplasmatales. Interspecies interactions, the presence of a unique internal tubular organelle [Comolli, et al. (2009) ISME J 3:159–167], and many genes previously only affiliated with Crenarchaea or Bacteria indicate extensive unique physiology in organisms that branched close to the time that Cren- and Euryarchaeotal lineages diverged.

Hydrothermal plumes are hot spots of microbial biogeochemistry in the deep ocean, yet little is known about the diversity or ecology of microorganisms inhabiting plumes. Recent biogeochemical evidence shows that Mn(II) oxidation in the Guaymas Basin (GB) hydrothermal plume is microbially mediated and suggests that the plume microbial community is distinct from deep-sea communities. Here we use a molecular approach to compare microbial diversity in the GB plume and in background deep seawater communities, and cultivation to identify Mn(II)-oxidizing bacteria from plumes and sediments. Despite dramatic differences in Mn(II) oxidation rates between plumes and background seawater, microbial diversity and membership were remarkably similar. All bacterial clone libraries were dominated by Gammaproteobacteria and archaeal clone libraries were dominated by Crenarchaeota. Two lineages, both phylogenetically related to methanotrophs and/or methylotrophs, were consistently over-represented in the plume. Eight Mn(II)-oxidizing bacteria were isolated, but none of these or previously identified Mn(II) oxidizers were abundant in clone libraries. Taken together with Mn(II) oxidation rates measured in laboratory cultures and in the field, these results suggest that Mn(II) oxidation in the GB hydrothermal plume is mediated by genome-level dynamics (gene content and/or expression) of microorganisms that are indigenous and abundant in the deep sea but have yet to be unidentified as Mn(II) oxidizers.

Microorganisms play important roles in mediating biogeochemical reactions in deep-sea hydrothermal plumes, but little is known regarding the mechanisms that underpin these transformations. At Guaymas Basin (GB) in the Gulf of California, hydrothermal vents inject fluids laden with dissolved Mn(II) (dMn) into the deep waters of the basin where it is oxidized and precipitated as particulate Mn(III/IV) oxides, forming turbid hydrothermal “clouds”. Previous studies have predicted extremely short residence times for dMn at GB and suggested they are the result of microbially-mediated Mn(II) oxidation and precipitation. Here we present biogeochemical results that support a central role for microorganisms in driving Mn(II) oxidation in the GB hydrothermal plume, with enzymes being the primary catalytic agent. dMn removal rates at GB are remarkably fast for a deep-sea hydrothermal plume (up to 2 nM hr-1). These rapid rates were only observed within the plume, not in background deep-sea water above the GB plume or at GB plume depths (~1750 – 2000 m) in the neighboring Carmen Basin, where there is no known venting. dMn removal is dramatically inhibited under anoxic conditions and by the presence of the biological poison, sodium azide. A conspicuous temperature optimum of dMn removal rates (~40°C) and a saturation-like (i.e. Michaelis-Menten) response to O2 concentration were observed, indicating an enzymatic mechanism. dMn removal was resistant to heat treatment used to select for spore-forming organisms, but very sensitive to low concentrations of added Cu, a cofactor required by the putative Mn(II) oxidizing enzyme. Extended X-ray absorption fine structure spectroscopy (EXAFS) and synchrotron radiation-based X-ray diffraction (SR-XRD) revealed the Mn oxides to have a hexagonal birnessite or δ-MnO2-like mineral structure, indicating that these freshly formed deep-sea Mn oxides are strikingly similar to primary biogenic Mn oxides produced by laboratory cultures of bacteria. Overall, these results reveal a vigorous Mn biogeochemical cycle in the GB hydrothermal plume, where a distinct microbial community enzymatically catalyzes rapid Mn(II) oxidation and the production of Mn biooxides.

Background. Analyses of DNA sequences from cultivated microorganisms have revealed genome-wide, taxa-specific nucleotide compositional characteristics, referred to as genome signatures. These signatures have far-reaching implications for understanding genome evolution and potential application in classification of metagenomic sequence fragments. However, little is known regarding the distribution of genome signatures in natural microbial communities or the extent to which environmental factors shape them. Results. We analyzed metagenomic sequence data from two acidophilic biofilm communities, including composite genomes reconstructed for nine archaea, three bacteria, and numerous associated viruses, as well as thousands of unassigned fragments from strain variants and low-abundance organisms. Genome signatures, in the form of tetranucleotide frequencies analyzed by emergent self-organizing maps, segregated sequences from all known populations sharing < 50-60% average amino acid identity and revealed previously unknown genomic clusters corresponding to low-abundance organisms and a putative plasmid. Signatures were pervasive genome wide. Clusters were resolved because intra-genome differences resulting from translational selection or protein adaptation to the intracellular (pH ~5) versus extracellular (pH ~1) environment were small relative to inter-genome differences. We found that these genome signatures stem from multiple influences but are primarily manifested through codon composition, which we propose is the result of genome-specific mutational biases. Conclusions. An important conclusion is that shared environmental pressures and interactions among coevolving organisms do not obscure genome signatures in acid mine drainage communities. Thus, genome signatures can be used to assign sequence fragments to populations, an essential prerequisite if metagenomics is to provide ecological and biochemical insights into the functioning of microbial communities.

We analyzed near-complete population (composite) genomic sequences for coexisting acidophilic iron-oxidizing Leptospirillum group II and III bacteria (phylum Nitrospirae) and an extrachromosomal plasmid from a Richmond Mine, Iron Mountain, CA, acid mine drainage biofilm. Community proteomic analysis of the genomically characterized sample and two other biofilms identified 64.6% and 44.9% of the predicted proteins of Leptospirillum groups II and III, respectively, and 20% of the predicted plasmid proteins. The bacteria share 92% 16S rRNA gene sequence identity and >60% of their genes, including integrated plasmid-like regions. The extrachromosomal plasmid carries conjugation genes with detectable sequence similarity to genes in the integrated conjugative plasmid, but only those on the extrachromosomal element were identified by proteomics. Both bacterial groups have genes for community-essential functions, including carbon fixation and biosynthesis of vitamins, fatty acids, and biopolymers (including cellulose); proteomic analyses reveal these activities. Both Leptospirillum types have multiple pathways for osmotic protection. Although both are motile, signal transduction and methyl-accepting chemotaxis proteins are more abundant in Leptospirillum group III, consistent with its distribution in gradients within biofilms. Interestingly, Leptospirillum group II uses a methyl-dependent and Leptospirillum group III a methyl-independent response pathway. Although only Leptospirillum group III can fix nitrogen, these proteins were not identified by proteomics. The abundances of core proteins are similar in all communities, but the abundance levels of unique and shared proteins of unknown function vary. Some proteins unique to one organism were highly expressed and may be key to the functional and ecological differentiation of Leptospirillum groups II and III.

Several closely related Mn(II)-oxidizing α-proteobacteria were isolated from very different marine environments: strain SI85-9A1 from the oxic/anoxic interface of a stratified Canadian fjord, strain HTCC 2156 from the surface waters off the Oregon coast, and strain AE01 from the dorsal surface of a hydrothermal vent tubeworm. 16S rRNA analysis reveals that these isolates are part of a tight phylogenetic cluster with previously characterized members of the genus Aurantimonas. Other organisms within this clade have been isolated from disparate environments such as surface waters of the Arctic and Mediterranean sea, a deep-sea hydrothermal plume, and a Caribbean coral. Further analysis of all these strains revealed that many of them are capable of oxidizing dissolved Mn(II) and producing particulate Mn(III/IV) oxides. Strains SI85-9A1 and HTCC 2156 were further characterized. Despite sharing nearly identical 16S rRNA gene sequences with the previously described Aurantimonas coralicida, whole genome DNA-DNA hybridization indicated that their overall genomic similarity is low. Polyphasic phenotype characterization further supported distinguishing characteristics among these bacteria. Thus SI85-9A1 and HTCC 2156 are described as two new species within the family ‘Aurantimionadaceae’: Aurantimonas manganoxydans sp. nov. and Aurantimonas litoralis sp. nov. This clade of bacteria is widely distributed around the globe and may be important contributors to Mn cycling in many environments. Our results highlight the difficulty in utilizing 16S rRNA-based approaches to investigate the microbial ecology of Mn(II) oxidation.

Microorganisms catalyze the formation of naturally occurring Mn oxides, but little is known about the biochemical mechanisms of this important biogeochemical process. We used tandem mass spectrometry to directly analyze the Mn(II)-oxidizing enzyme from marine Bacillus spores, identified as a Mn oxide band with an in-gel activity assay. Nine distinct peptides recovered from the Mn oxide band of two Bacillus species were unique to the multicopper oxidase MnxG, and one peptide was from the small hydrophobic protein MnxF. No other proteins were detected in the Mn oxide band, indicating that MnxG (or a MnxF/G complex) directly catalyzes biogenic Mn oxide formation. The Mn(II) oxidase was partially purified and found to be resistant to many proteases and active even at high concentrations of sodium dodecyl sulfate. Comparative analysis of the genes involved in Mn(II) oxidation from three diverse Bacillus species revealed a complement of conserved Cu-binding regions not present in well-characterized multicopper oxidases. Our results provide the first direct identification of a bacterial enzyme that catalyzes Mn(II) oxidation and suggest that MnxG catalyzes two sequential one-electron oxidations from Mn(II) to Mn(III) and from Mn(III) to Mn(IV), a novel type of reaction for a multicopper oxidase.

Microbial Mn(II) oxidation has important biogeochemical consequences in marine, freshwater, and terrestrial environments, but many aspects of the physiology and biochemistry of this process remain obscure. Here, we report genomic insights into Mn(II) oxidation by the marine alphaproteobacterium Aurantimonas sp. Strain SI85-9A1, isolated from the oxic/anoxic interface of a stratified fjord. The SI85-9A1 genome harbors the genetic potential for metabolic versatility, with genes for organoheterotrophy, methylotrophy, oxidation of sulfur and carbon monoxide, the ability to grow over a wide range of O2 concentrations (including microaerobic conditions), and the complete Calvin cycle for carbon fixation. Although no growth could be detected under autotrophic conditions with Mn(II) as the sole electron donor, cultures of SI85-9A1 grown on glycerol are dramatically stimulated by addition of Mn(II), suggesting an energetic benefit from Mn(II) oxidation. A putative Mn(II) oxidase is encoded by duplicated multicopper oxidase genes that have a complex evolutionary history including multiple gene duplication, loss, and ancient horizontal transfer events. The Mn(II) oxidase was most abundant in the extracellular fraction, where it cooccurs with a putative hemolysin-type Ca2_- binding peroxidase. Regulatory elements governing the cellular response to Fe and Mn concentration were identified, and 39 targets of these regulators were detected. The putative Mn(II) oxidase genes were not among the predicted targets, indicating that regulation of Mn(II) oxidation is controlled by other factors yet to be identified. Overall, our results provide novel insights into the physiology and biochemistry of Mn(II) oxidation and reveal a genome specialized for life at the oxic/anoxic interface.

Manganese (Mn), the fifth most abundant element in the Earth’s crust, and after iron (Fe) the most abundant transitional metal, is an essential micronutrient for most organisms. Found at the core of the reaction center of the light-harvesting complex, MN is particularly important for plants and other photosynthetic microorganisms. It is also a cofactor for numerous enzymes, including manganese superoxide dismutase, manganese catalase, and manganese-dependent ribonucleotide reductase (36). The redox properties of Mn make it central to a variety of biological processes and result in significant and often rapid biogeochemical cycling that is mediated by abiotic and biotic oxidation and reduction, biological uptake, and mineral formation. For overviews on Mn, the reader is referred to recent review articles (35, 69, 113, 117).

Microbial oxidation and precipitation of manganese at deep-sea hydrothermal vents are important oceanic biogeochemical processes, yet nothing is known about the types of microorganisms or mechanisms involved. Here we report isolation of a number of diverse spore-forming Mn(II)-oxidizing Bacillus species from Guaymas Basin, a deep-sea hydrothermal vent environment in the Gulf of California, where rapid microbially mediated Mn(II) oxidation was previously observed. mnxG multicopper oxidase genes involved in Mn(II) oxidation were amplified from all Mn(II)-oxidizing Bacillus spores isolated, suggesting that a copper-mediated mechanism of Mn(II) oxidation could be important at deep-sea hydrothermal vents. Phylogenetic analysis of 16S rRNA andmnxG genes revealed that while many of the deep-sea Mn(II)-oxidizing Bacillus species are very closely related to previously recognized isolates from coastal sediments, other organisms represent novel strains and clusters. The growth and Mn(II) oxidation properties of these Bacillus species suggest that in hydrothermal sediments they are likely present as spores that are active in oxidizing Mn(II) as it emerges from the seafloor.

Webb, S. M., et al. (2005), Evidence for the presence of Mn(III) intermediates in the bacterial oxidation of Mn(II), Proceedings of the National Academy of Sciences of the United States of America, 102(15), 5558-5563.

Bacterial oxidation of Mn(II) to Mn(IV) is believed to drive the oxidative segment of the global biogeochemical Mn cycle and regulates the concentration of dissolved Mn(II) in the oceanic water column, where it is a critical nutrient for planktonic primary productivity. Mn(II) oxidizing activity is expressed by numerous phylogenetically diverse bacteria and fungi, suggesting that it plays a fundamental and ubiquitous role in the environment. This important redox system is believed to be driven by an enzyme or enzyme complex involving a multicopper oxidase, although the biochemical mechanism has never been conclusively demonstrated. Here, we show that Mn(II) oxidation by spores of the marine Bacillus sp. strain SG-1 is a result of two sequential one-step electron transfer processes, both requiring the putative multicopper oxidase, MnxG, in which Mn(III) is a transient intermediate. A kinetic model of the oxidation pathway is presented, which shows that the Mn(II) to Mn(III) step is the rate-limiting step. Thus, oxidation of Mn(II) appears to involve a unique multicopper oxidase system capable of the overall two-electron oxidation of its substrate. This enzyme system may serve as a source for environmental Mn(III), a strong oxidant and competitor for siderophorebound Fe(III) in nutrient-limited environments. That metabolically dormant spores catalyze an important biogeochemical process intimately linked to the C, N, Fe, and S cycles requires us to rethink the role of spores in the environment.

Manganese(IV) oxides produced through microbial activity, i.e., biogenic Mn oxides or Mn biooxides, are believed to be the most abundant and highly reactive Mn oxide phases in the environment. They mediate redox reactions with organic and inorganic compounds and sequester a variety of metals. The major pathway for bacterial Mn(II) oxidation is enzymatic, and although bacteria that oxidize Mn(II) are phylogenetically diverse, they require a multicopper oxidase-like enzyme to oxidize Mn(II). The oxidation of Mn(II) to Mn(IV) occurs via a soluble or enzyme-complexed Mn(III) intermediate. The primary Mn(IV) biooxide formed is a hyllomanganate most similar to ±-MnO2 or acid birnessite. Metal sequestration by the Mn biooxides occurs predominantly at vacant layer octahedral sites.